The Complete Mitochondrial Genomes Of Three Papilionid Butterfly Species And Their Relevant Phylogenetic Implications

As a group of large and beautiful butterflies, the Papilionidae(Insecta:Lepidoptera) includes three subfamilies, namely Baroniinae, Papilioninae,Parnassiinae. A total of about 600 papilionid species are distributed all over the world, among which 129 species exist in China. However, up to the present only 13 complete mitogenome sequences of the Papilionidae butterflies have been reported.The monophyly of Papilionidae has been continuously confirmed, and supported by morphological and molecular evidences. Nonetheless, many problems concerning the phylogeny of Papilionidae, especially about the interordinal relationships among the main Papilionidae lineages, are still standing controversial and have not reached the same view as far as the morphological characteristics, molecular data and palaeontological evidences are concerned.With the purpose of exploring the evolutionary path diagram and timescale in butterfly species, we sequenced the complete mitochondrial genomes(mitogenomes) of three papilionid(Byasa alcinous, Pazala timur and Parnassius apollo) by the way of using short/long PCR and primer-walking technologies. We made a detailed analysis in the gene organization and variation characteristics, by combining with the corresponding sequence data in other available butterfly mitogenomes. Meanwhile, the phylogenic relationship analysis and the divergence time estimation of the Papilionidae were carried out based on the calibrated fossil records using various phylogenic and relaxed molecular clock dating methods, in order to sheld new insights into the evolutionary studies of the Papilionidae.Results indicate that the whole mitogenomes of B. alcinous, P. timur and P.apollo are 15, 266 bp, 15, 226 bp and 15, 404 bp in length, respectively, and they all contained 22 transfer RNA genes(t RNAs), 13 protein-coding genes(PCGs), 2ribosomal RNA genes(rRNAs) and one control region(A+T-rich region). A small number of intergenic overlapping sequences and spacer sequences are scattered allover the entire mitogenomes, with the number of overlapping spacer sequences and intergenic sequences ranging from 10~12 and 10~14, the total length ranging from26~30 bp, and 73~164 bp, respectively. Except COI gene uses CGA as its initiation codon, All the PCGs start with ATN. The COII and COI genes in these three butterfly mitogenomes all use single T as their termination codons. Moreover, the ND4 gene in B. alcinous and ND3 gene in P. timur also terminate with single T.Other PCGs use TAA or TAG as their classic termination codons. Each of the non-coding A+T-region is composed mostly of non-repetitive sequences, but harbors some typical structures characteristic of lepidopterans: the motif ATAGA followed by a poly-T stretch and a microsatellite-like repeat(TA)n preceded by the ATTTA motif.The obtained consensus phylogenetic trees of the butterflies indicated that the interfamial relationship within the butterflies was:(((((Riodinidae + Lycaenidae) +Nymphalidae) + Pieridae) + Hesperiidae) + Papilionidae). Simultaneously, the tribe relationship within the subfamily Papilioninae was:(((Papilionini + Teinopalpini) +Troidini) + Lampropterini). The phylogenetic analysis also indicated that the two tribes of the subfamily Parnassiinae(Parnassiini and the Zerynthiini) were sister groups to each other. The phylochronological analysis showed that the divergence of the Papilionidae occurred during the late Cretaceous(estimated mean: 83.6 Ma,0.95 confidence interval: 68.9~98.3 Ma, 0.95%IC), while the Papilioninae and Parnassiinae occurred during the late Cretaceous(78.7 Ma; 63.3~94.1 Ma,0.95%IC) and the Palaeocene(57.1 Ma; 44.1~70.1 Ma, 0.95%IC), respectively.